HRP20192353T1 - Method of determining pik3ca mutational status in a sample - Google Patents

Method of determining pik3ca mutational status in a sample Download PDF

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HRP20192353T1
HRP20192353T1 HRP20192353TT HRP20192353T HRP20192353T1 HR P20192353 T1 HRP20192353 T1 HR P20192353T1 HR P20192353T T HRP20192353T T HR P20192353TT HR P20192353 T HRP20192353 T HR P20192353T HR P20192353 T1 HRP20192353 T1 HR P20192353T1
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Evrykleia LIANIDOU
Athina Markou
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Pharmassist Ltd
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Claims (18)

1. Metoda analiziranja prisutnosti DNK mutiranog alela PIK3CA u DNK uzorku, navedena metoda koja se sastoji od koraka izvođenja asimetrične lančane reakcije polimeraze (PCR) i lančane reakcije polimeraze (PCR) specifične za alelu navedenog DNK mutiranog alela; i izvođenje analize taljenja DNK proizvedenog u PCR-u, gdje se navedena lančana reakcija polimeraze (PCR) provodi upotrebom - početnice specifične za mutirani alel zajedno s 3’ (tri početnice) kraja niza DNK mutiranog alela koji će se umnožiti; navedena početnica specifična za mutirani alel sastoji se od mutirane strane i nepodudarnosti odgovarajućeg DNK divljeg tipa, i - neoznačene sonde za blokiranje da je oligonukleotid zajedno sa sekvencom divljeg tipa prvog niza DNK divljeg tipa koji odgovara prvom nizu DNK mutiranog alela na odgovarajućem mjestu, na kojem je prisutna mutacija koja se mora detektirati; navedena neoznačena sonda sastoji se od dodatne nepodudarnosti u odnosu na prvi niz DNK mutiranog alela osim strane mutacije, i od sonde koja je blokirana jer djeluje kao početnica za DNK sintezu u reakciji PCR, i - uobičajene početnice koja služi kao dopuna 3' kraja drugog niza DNK mutiranog alela koji će se umnožiti u reakciji PCR, i u sklopu koje se analiza taljenja provodi upotrebom - sonde za taljenje koja je neoznačena sonda koja je oligonukleotid, koji se sastoji od sekvence koja služi kao dopuna DNK alela divljeg tipa i preklapa se sa sekvencom DNK mutiranog alela, i - detektabilne komponente za mjerenje temperature taljenja komponenti DNK s duplim nizom na najmanje uključenoj komponenti s duplim nizom sonde za taljenje vezane s umnoženim nizom DNK mutiranog alela ili niza DNK divljeg alela, u sklopu čega se temperatura taljenja razlikuje između komponente s duplim nizom sonde za taljenje vezane na umnoženi niz mutiranog alela i sonde za taljenje vezane na umnoženi niz divljeg alela.1. A method of analyzing the presence of PIK3CA mutated allele DNA in a DNA sample, said method consisting of the steps of performing asymmetric polymerase chain reaction (PCR) and polymerase chain reaction (PCR) specific for the allele of said DNA mutated allele; and performing melting analysis of the PCR-produced DNA, wherein said polymerase chain reaction (PCR) is carried out using - primers specific for the mutated allele together with the 3' (three primers) end of the DNA sequence of the mutated allele that will be amplified; said primer specific for the mutated allele consists of the mutated side and a mismatch of the corresponding wild-type DNA, and - unlabeled probes for blocking that the oligonucleotide together with the wild-type sequence of the first DNA sequence of the wild-type corresponding to the first DNA sequence of the mutated allele is in the appropriate place, where the mutation to be detected is present; said unlabeled probe consists of an additional mismatch with respect to the first DNA strand of the mutated allele other than the foreign mutation, and of a probe that is blocked because it acts as a primer for DNA synthesis in the PCR reaction, and - the usual primers that serve as complements to the 3' end of the second DNA sequence of the mutated allele that will be amplified in the PCR reaction, and within which the melting analysis is performed using - a melting probe that is an unlabeled probe that is an oligonucleotide, consisting of a sequence that serves as a complement to the DNA of the wild-type allele and overlaps with the DNA sequence of the mutated allele, and - detectable components for measuring the melting temperature of the double-stranded DNA component on the least included double-stranded component of the melting probe linked to the amplified DNA sequence of the mutated allele or the DNA sequence of the wild-type allele, within which the melting temperature differs between the double-stranded component of the melting probe linked to the amplified sequence of the mutated allele and melting probes linked to the amplified sequence of the wild-type allele. 2. Metoda u skladu sa zahtjevom 1, u sklopu koje se DNK mutiranog alela umnožava tijekom reakcije PCR, sastoji se od egzona 9 (ID BR. SEKV.: 1) i/ili egzona 20 ID BR. SEKV.: 2) od PIK3CA, a sekvenca specifične početnice mutiranog alela komplementarna je DNK nizu egzona 9 (ID BR. SEKV.: 4) ili 20 (ID BR. SEKV.: 9).2. The method according to claim 1, in which the DNA of the mutated allele is amplified during the PCR reaction, consisting of exon 9 (SEQ ID NO: 1) and/or exon 20 ID NO. SEQ ID NO: 2) of PIK3CA, and the specific primer sequence of the mutated allele is complementary to the DNA sequence of exon 9 (SEQ ID NO: 4) or 20 (SEQ ID NO: 9). 3. Metoda u skladu sa zahtjevom 1 ili 2, u sklopu koje je sonda za taljenje neoznačena sonda za blokiranje.3. The method of claim 1 or 2, wherein the melting probe is an unlabeled blocking probe. 4. Metoda u skladu s bilo kojim spomenutim prethodnim zahtjevima, u sklopu koje neoznačena sonda za blokiranje ima modificirani 3'-kraj koji je modificiran dodanom fosfatnom grupom u usporedbi s PCR početnicom za umnožavanje.4. A method according to any of the preceding claims, wherein the unlabeled blocking probe has a modified 3'-end which is modified by an added phosphate group compared to the PCR amplification primer. 5. Metoda u skladu s bilo kojim spomenutim prethodnim zahtjevima, u sklopu koje se detektabilna komponenta sastoji od fluorescentne komponente i u sklopu kojih analiza taljenja uključuje detekciju fluorescentne komponente.5. A method according to any of the preceding claims, wherein the detectable component comprises a fluorescent component and wherein the melting analysis includes detection of the fluorescent component. 6. Metoda u skladu sa zahtjevom 5, u sklopu koje je fluorescentna komponenta fluorescentna boja koja se veže na DNK, grupe koja se sastoji od boja LC-Green Plus ili SYBR Green I, a koje emitiraju fluorescenciju samo u prisutnosti DNK s duplim nizom u uzorku.6. The method according to claim 5, in which the fluorescent component is a DNA-binding fluorescent dye of the group consisting of LC-Green Plus or SYBR Green I dyes, which emit fluorescence only in the presence of double-stranded DNA in sample. 7. Metoda u skladu s bilo kojim spomenutim prethodnim zahtjevima, u sklopu koje su neoznačena sonda za blokiranje i uobičajena početnica prisutni u visokim koncentracijama nego u početnici specifičnoj za mutirani alel.7. A method according to any of the preceding claims, wherein the unlabeled blocking probe and the common primer are present in higher concentrations than in the primer specific for the mutated allele. 8. Metoda u skladu s bilo kojim spomenutim prethodnim zahtjevima, u sklopu koje je mutacija prisutna u egzonu 9 od PIK3CA, a početnica specifična za mutirani alel sastoji se od sekvence 5’-TTTCTCCTGATT-3’ (ID BR. SEKV.: 3) ili je 5’- ACTCCATAGAAAATCTTTCTCCTGATT-3’ (ID BR. SEKV.: 4), u kojoj se T odnosi na stranu mutacije, a A na dodatnu nepodudarnost.8. A method according to any of the preceding claims, wherein the mutation is present in exon 9 of PIK3CA, and the primer specific for the mutated allele consists of the sequence 5'-TTTCTCCTGATT-3' (SEQ ID NO: 3) or is 5'- ACTCCATAGAAAATCTTTCCTCCTGATT-3' (SEQ ID NO: 4), in which T refers to the side of the mutation and A to the additional mismatch. 9. Metoda u skladu sa zahtjevom 8, u sklopu koje se neoznačena sonda za blokiranje sastoji od sekvence 5’-CTGATCAGTGA-3’ (ID BR. SEKV.: 5) i PCR blokirajuća komponenta se sastoji od sekvence 5’- CTTTCTCCTGATCAGTGATTTCAGAG-P-3’ (ID BR. SEKV.: 6), u kojoj se P odnosi na fosfat, a A na dodatnu nepodudarnost.9. The method according to claim 8, in which the unlabeled blocking probe consists of the sequence 5'-CTGATCAGTGA-3' (SEQ ID NO: 5) and the PCR blocking component consists of the sequence 5'- CTTTCTCCTGATCAGTGATTTCAGAG-P -3' (SEQ ID NO: 6), wherein P refers to a phosphate and A to an additional mismatch. 10. Metoda u skladu sa zahtjevima 8 ili 9, u sklopu koje je uobičajena početnica 75 % do 100 % identična u odnosu na sekvenci 5’-GCTCAAAGCAATTTCTACACGAGA-3’ (ID BR. SEKV.: 7).10. The method according to claims 8 or 9, in which the common primer is 75% to 100% identical to the sequence 5'-GCTCAAAGCAATTTCTACACGAGA-3' (SEQ ID NO.: 7). 11. Metoda u skladu s jednim od zahtjeva od 1 do 7, u sklopu koje je mutacija prisutna u egzonu 20 (ID BR. SEKV.: 2) od PIK3CA, i u sklopu koje se početnica specifična za mutirani alel sastoji od sekvence 5’-AATGATGCACG -3’ (ID BR. SEKV.: 8) u kojoj se G odnosi na stranu mutacije.11. The method according to one of claims 1 to 7, in which the mutation is present in exon 20 (SEQ ID NO: 2) of PIK3CA, and in which the primer specific for the mutated allele consists of the sequence 5'- AATGATGCACG -3' (SEQ ID NO: 8) in which G refers to the side of the mutation. 12. Metoda u skladu sa zahtjevom 1, u sklopu koje se početnica specifična za mutirani alel sastoji od 5’-ATGAAACAAATGAATGATGCACG-3’ (ID BR. SEKV.: 9) u kojoj se G odnosi na stranu mutacije.12. The method according to claim 1, in which the primer specific for the mutated allele consists of 5'-ATGAAACAAATGAATGATGCACG-3' (SEQ ID NO: 9) in which G refers to the side of the mutation. 13. Metoda u skladu sa zahtjevima 11 ili 12, u sklopu koje se neoznačena sonda za blokiranje sastoji od sekvence 5’-TGCACATCATG-3’ (ID BR. 5 SEKV.: 10) i PCR blokirajuće komponente ili sekvence 5’-GAATGATGCACATCATGGTGG-P-3’ (SEQ ID NO: 11) u kojoj se P odnosi na fosfat. 13. The method according to claims 11 or 12, in which the unlabeled blocking probe consists of the sequence 5'-TGCACATCATG-3' (SEQ ID NO: 5: 10) and the PCR blocking component or sequence 5'-GAATGATGCACATCATGGTGG- P-3' (SEQ ID NO: 11) wherein P refers to phosphate. 14. Metoda u skladu s bilo kojim od zahtjeva 10, u sklopu koje je uobičajena početnica 75 % do 100 % identična u odnosu na sekvencu 5’-TCTCAGTTATCTTTTCAGTTCAATGC-3’ (ID BR. SEKV.: 12).14. The method according to any one of claims 10, wherein the common primer is 75% to 100% identical to the sequence 5'-TCTCAGTTATCTTTTCAGTTCAATGC-3' (SEQ ID NO: 12). 15. Metoda za i) dijagnosticiranje maligne neoplastične bolesti na subjektu, i/ili ii) predviđanje efikasnosti tretmana maligne neoplastične bolesti na subjektu, i/ili iii) procjenu ishoda tretmana maligne neoplastične bolesti na subjektu, i/ili iv) procjenu ponovne pojave maligne neoplastične bolesti na subjektu u sklopu koje je subjekt sisavac koji ima, ili se sumnja da ima, malignu neoplastičnu bolest, u sklopu čega se navedena metoda sastoji od analiziranja prisutnosti DNK mutiranog alela PIK3CA u uzorku u skladu s metodom zahtjeva 1od 1 do 14.15. Method for i) diagnosing a malignant neoplastic disease on the subject, and/or ii) predicting the effectiveness of the treatment of malignant neoplastic disease on the subject, and/or iii) assessment of the outcome of the treatment of malignant neoplastic disease on the subject, and/or iv) assessment of recurrence of malignant neoplastic disease in the subject in which the subject is a mammal that has, or is suspected of having, a malignant neoplastic disease, as part of which the mentioned method consists of analyzing the presence of DNA mutated PIK3CA allele in the sample in accordance with the method of claim 1 from 1 to 14. 16. Metoda u skladu sa zahtjevom 15, u sklopu koje je uzorak biološki uzorak dobiven od subjekta.16. The method according to claim 15, wherein the sample is a biological sample obtained from the subject. 17. Metoda u skladu sa zahtjevom 15 ili 16, u sklopu koje je maligna neoplastična bolest rak dojke, rak debelog crijeva, rak pluća, rak vrata maternice, rak jajnika, rak jednjaka, rak na mozgu, rak kože, rak jetre, rak gušterače, rak glave i vrata, rak želuca i rak štitnjače.17. The method according to claim 15 or 16, in which the malignant neoplastic disease is breast cancer, colon cancer, lung cancer, cervical cancer, ovarian cancer, esophageal cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer , head and neck cancer, stomach cancer and thyroid cancer. 18. Komplet za i) detekciju prisutnosti mutacije u PIK3CA u uzorku, i/ili ii) detekciju maligne neoplastične bolesti na subjektu, ili iii) dijagnosticiranje maligne neoplastične bolesti na subjektu, ili iv) predviđanje ishoda tretmana maligne neoplastične bolesti na subjektu, ili v) procjenu efikasnosti tretmana maligne neoplastične bolesti na subjektu, ili vi) procjenu ponovne pojave maligne neoplastične bolesti na subjektu; navedeni komplet sastoji se od - prvog polinukleotida koji se sastoji od barem sekvence 5’-TTTCTCCTGATT-3’ (ID BR. SEKV.: 3), ili se sastoji od barem sekvence 5’-ACTCCATAGAAAATCTTTCTCCTGATT-3’ (ID BR. SEKV.: 4), u kojoj se T odnosi na stranu mutacije, a A na dodatnu nepodudarnost, i - drugog polinukleotida koji se sastoji od barem sekvence 5’- AATGATGCACG -3’ (ID BR. SEKV.: 8), ili se sastoji od barem sekvence 5’-TGAAACAAATGAATGATGCACG-3’ (ID BR. SEKV.: 9), u kojoj se G odnosi na stranu mutacije, i - trećeg polinukleotida koji se sastoji od barem sekvence 5’-CTGATCAGTGA-3’ (ID BR. SEKV.: 5) i PCR blokirajuće komponente ili koji se sastoji od barem sekvence 5’-CTTTCTCCTGATCAGTGATTTCAGAG-P-3’ (ID BR. SEKV.: 6), i u kojoj se P odnosi na fosfat, a A na dodatnu nepodudarnost, i - četvrtog polinukleotida koji se sastoji od barem sekvence 5’-TGCACATCATG-3’ (ID BR. SEKV.: 10) i PCR blokirajuće komponente, ili se sastoji od barem sekvence 5’-GAATGATGCACATCATGGTGG-P-3’ (ID BR. SEKV.: 11), i u kojoj se P odnosi na fosfat, i - petog polinukleotida koji je 75 % do 100 % identičan sekvenci 5’-GCTCAAAGCAATTTCTACACGAGA-3’ (ID BR. SEKV.: 7), i - šestog polinukleotida koji je 75 % do 100 % identičan sekvenci 5’-TCTCAGTTATCTTTTCAGTTCAATGC-3’ (ID BR. SEKV.: 12).18. Kit for i) detection of the presence of a mutation in PIK3CA in the sample, and/or ii) detection of malignant neoplastic disease on the subject, or iii) diagnosing a malignant neoplastic disease on the subject, or iv) predicting the outcome of the treatment of malignant neoplastic disease on the subject, or v) assessment of the effectiveness of the treatment of malignant neoplastic disease on the subject, or vi) assessment of recurrence of malignant neoplastic disease in the subject; the said set consists of - the first polynucleotide consisting of at least the sequence 5'-TTTCTCCTGATT-3' (SEQ ID NO.: 3), or consisting of at least the sequence 5'-ACTCCATAGAAAATCTTTCTCCTGATT-3' (SEQ ID NO.: 4), in where T refers to the side of the mutation and A to the additional mismatch, i - another polynucleotide consisting of at least the sequence 5'- AATGATGCACG -3' (SEQ ID NO.: 8), or consisting of at least the sequence 5'-TGAAACAAATGAATGATGCACG-3' (SEQ ID NO.: 9), in to which G refers to the mutation side, i - a third polynucleotide consisting of at least the sequence 5'-CTGATCAGTGA-3' (SEQ ID NO.: 5) and a PCR blocking component or consisting of at least the sequence 5'-CTTTCTCCTGATCAGTGATTTCAGAG-P-3' (SEQ ID NO. .: 6), and in which P refers to phosphate and A to an additional mismatch, i - the fourth polynucleotide consisting of at least the sequence 5'-TGCACATCATG-3' (SEQ ID NO: 10) and a PCR blocking component, or consisting of at least the sequence 5'-GAATGATGCACATCATGGTGG-P-3' (SEQ ID NO: .: 11), and in which P refers to phosphate, i - the fifth polynucleotide which is 75% to 100% identical to the sequence 5'-GCTCAAAGCAATTTCTACACGAGA-3' (SEQ ID NO.: 7), and - the sixth polynucleotide which is 75% to 100% identical to the sequence 5'-TCTCAGTTATCTTTTCAGTTCAATGC-3' (SEQ ID NO.: 12).
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